Macrophage lipid chemoattractant

ABSTRACT

This invention encompasses a substantially homogeneous lipid chemoattractant released from stressed mammalian tissue which is a neutral lipid which is acid labile and stable to base and is stable in boiling water. This lipid recruits macrophages but not neutrophils to stressed tissue. The invention also encompasses a method for detecting injured tissue by detecting the presence of the above described lipid chemoattractant in body fluids such as urine, serum and saliva. The invention also includes a method for reducing recruitment of macrophages to injured tissue by reducing the amount of the above lipid chemoattractant or by blocking the interaction of this lipid chemoattractant with its macrophage binding site. The addition of this lipid chemoattractant to injured skin tissue promotes healing.

This application is a divisional of application Ser. No. 08/470,974filed Jun. 6, 1995, which in turn is now U.S. Pat. No. 5,663,450, and acontinuation-in-part of U.S. patent application Ser. No. 08/107,958filed Aug. 17, 1993, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of The Invention

This invention is in the field of chemotaxis and chronic inflammation.

2. Related Prior Art

The effector cell of chronic inflammation is the macrophage. Themacrophage in inflamed tissues is derived from circulating monocytesthat originate in the bone marrow. In response to signals from injuredtissue, as yet incompletely understood, the monocyte binds to and thenmigrates beneath the endothelium lining the vascular structures throughwhich it is circulating. Once in a subendothelial position, the monocyteactivates and differentiates into a macrophage. The activated macrophagereleases a variety of factors that degrade extracellular matrix,stimulate collagen production, and promote proliferation of endothelialcells, fibroblasts, and vascular smooth muscle cells. These factorsinclude proteases, reactive oxygen species, and cytokines such asinterleukin-1, tumor necrosis factor, and platelet-derived growthfactors, among others, Nathan, C. F. 1987. J. Clin. Invest. 79: 319-326.The end result is the promotion of wound healing or, in the case ofinflammation occurring within a bodily organ, cell proliferation andsclerosis that may eventuate in impairment of organ function. Thus themacrophage is central to the pathological processes underlyingmyocardial fibrosis, atherosclerosis, restenosis, pulmonary fibrosis,progressive nephrosclerosis, arthritis, and inflammatory bowel disease,to cite a few examples of chronic inflammatory conditions, Kissane, J.ed. Anderson's Pathology, 1990. C. V. Mosby, St. Louis, Mo. pp. 89-96,615-730, 804-871, 920-1047, 1153-1199, 2065-2105.

There are two components of monocyte movement into tissues. The firstcomponent consists of monocyte adhesion to the endothelium. There is agrowing literature on the expression of adhesion proteins by endothelialand other cells that promote the binding of leukocytes, includingmonocytes, to the endothelium, Springer, T. 1990. Nature, 346: 425-433.The second component is the signal for chemotaxis, the signal releasedby traumatized tissue that induces migration of the monocyte beyond theendothelial cell to which it is adhering and into the tissue underlyingthe endothelium.

The factors regulating the subendothelial migration of monocytes intotissue in normal and inflammatory states are not well understood. Likeneutrophils, monocytes display chemotactic migration to C5a, thebacterial tripeptide f-Met-Leu-Phe, proteolytic fragments of collagenand fibronectin, platelet-derived growth factor, transforming growthfactor-β, and neuropeptides. Schiffman, E., & Gallin, J. 1979. Curr. TopCell Reg. 15: 203-213; Pierce, G., et al 1989. J. Cell Biol. 109: 429;Snyderman, R., & Mergenhagen S. 1976. In Immunobiology of the Macrophagepp. 323-348, New York Academic Press.

The contribution of the factors cited above to maintaining themononuclear leukocytic invasion of chronically inflamed tissues has notbeen defined. Since these factors attract both neutrophils andmonocyte/macrophages, and because neutrophils are not a prominentcomponent of chronic inflammatory lesions, there is continuing interestin further identifying chemotactic signals unique to the monocyte ormacrophage. The identification of such a chemotactic agent should meetthree criteria: 1) it should be chemotactic for monocytes but notneutrophils in vitro; 2) it should be identified in lesions associatedwith chronic infiltration by monocytes/macrophages; and 3) interruptionof its synthesis, release, or receptor activation should be associatedwith amelioration of chronic inflammation in vivo.

There is only one known chemotactic signal, a protein, unique to themonocyte. It is a lymphocyte-derived, 8 kD, chemotactic protein known asmacrophage chemotactic protein, which has been sequenced, Furutani, Y.,et al 1989. Biochim. Biophys. Res. Comm. 159: 249. However, no in vivoinhibition of its function has been achieved to date to confirm its rolein macrophage migration in vivo.

It has become evident that altered lipid metabolism, particularlyhyperlipidemia, may induce or augment monocyte migration into the wallsof vascular tissue. The early phase of atherosclerosis is characterizedby subendothelial migration of monocytes in the aorta and coronaryarteries, Ross, R. 1986. N. Eng. J. Med 296: 488. Hyperlipidemiaaccelerates the renal infiltration by monocytes in chronic inflammationof the glomerulus and interstitium, Diamond, J., & Karnovsky, M. 1988.Kid. Int. 33: 917. Although there is a clear association betweenlipidemia and monocyte migration into extravascular spaces, no lipidchemotactic factors have been described that are specific for monocytesat physiological concentrations. LTB₄ is highly chemotactic forpolymorphonuclear leukocytes (PMN), much less so for human monocytes,and not all for mouse or rat monocytes, Kreisle, R., et al 1985. J.Immunol. 134: 3356. Platelet activating factor is minimally chemotacticfor rat monocytes and more stimulatory for neutrophils, Goetzl, E., etal 1980. Biochim. Biophys. Res. Comm. 94: 881; Rovin, B., et al J.Immunol. A lipid generated during oxidative modification of lipoprotein,lysophosphatidyl choline, possesses modest chemotactic properties forhuman monocytes, Quinn, M., et al 1988. Proc. Natl. Acad. Sci. 85: 2805,but at extremely high concentrations not found in nature.

To date, there has been no demonstration that in vivo inhibition ofchemotactic factors specifically interrupts monocyte migration intotissues. This is true for both the macrophage chemotactic protein aswell as for lipid mediators of inflammation.

The model that we have most commonly employed for the study of monocytechemotaxis in vivo is nephrotoxic serum nephritis, in which rats receivean injection of rabbit anti-glomerular membrane (GBM) antibody.Polymorphonuclear leukocytes (PMNs) enter the glomerulus in the first 3hours followed by monocytes at 12-24 hours. Schreiner, G., et al 1978.J. Exp. Med. 147: 369. It has been shown that infiltration is notdependent upon complement activation. Schreiner, G., et al 1984, Lab.Invest. 51: 524.

The role of essential fatty acid (EFA) deficiency on the renalinfiltration by monocytes of these cells has been studied. Hurd, E., et.al. 1981. J. Clin. Invest. 67: 476, found that NZB/NZW F1 mice withsystemic lupus crythematosus did not die of renal failure if placed on adiet deficient in the essential fatty acids, linoleate and arachidonate,despite documented deposits of immune complexes in their glomeruli, andcirculating immune complexes. Subsequently it was shown thatEFA-deficiency resulted in a marked reduction in the number of residentrenal glomerular and interstitial macrophages Lefkowith, J., &Schreiner, G. 1987. J. Clin. Invest. 80: 947. When animals wereselectively repleted with (N-6) fatty acid supplementation, it wasobserved that a spontaneous macrophage repopulation of the glomerulusoccurred. It had been previously shown that resident macrophages in thekidney expressing the Ia+ antigene are highly stimulatory in a mixedlymphocyte culture reaction. The effect of whether depletion of thesecells from the kidney via this dietary manipulation would decrease theimmunogenicity of the kidney when transplanted across a majorhistocompatibility barrier was studied. Kidneys harvested from a LewisEFAD donor and transplanted into a Buffalo strain rat on a normal dietsurvived as allografts in the absence of immunosuppression of therecipient Schreiner, G., et al 1988. Science 240: 1032. Allografts fromEFAD donors normalized their lipid composition within the allogeneicrecipient and were repopulated with host macrophages within 5 days. Therapid repopulation of the kidney with host macrophages closelyparalleled the restoration of the essential fatty acid content ofnospholipids, suggesting that the seeding of organs by macrophages couldbe dependent in part upon a lipid pathway.

The potential role of this lipid pathway in mediating the inflammatoryinflux of macrophages into the kidney, in acute nephrotoxic nephritis,has been evaluated Schreiner, G., et al 1989. J. Immunol. 143: 3192. Theeffects of EFA-deficiency were striking. EFA-deficiency completelyprevented the influx of macrophages into the glomerulus during thecourse of the nephritis. In contrast, the preceding PMN influx wasunaffected. EFA-deficiency completely prevented polyuria, azotemia, andsodium retention, and largely abrogated the proteinuria. EFA-deficientmacrophages are not impaired in their ability to move chemotacticallytoward either C5a or platelet activating factor; and no circulatinginhibitors of chemotaxis were found in EFA-deficient serum, suggestingthe effect of EFA-deficiency may be exerted at the level oftissue-derived factors inducing monocyte migration Schreiner, G., et al1989. J. Immunol. 143: 3192; Rovin, B., et al 1990. J. Immunol 145:1238.

Using our dietary model, Diamond, J., et al 1989. Am. J. Physiol. 257:F798, discovered that animals deficient in essential fatty acids,deficient only during the period of acutely induced nephrotic state byPAN, were protected against the development of glomerular sclerosis fourmonths later. The protection against glomerular sclerosis did notcorrelate with the degree of proteinuria, hyperlipidemia, orhypertension. Rather, it directly correlated with the inhibition ofglomerular macrophage accumulation normally induced by the hyperlipemicstate of nephrosis.

It has been observed that EFA-deficiency also prevents the renalmononuclear cell interstitial infiltrate of acute PAN-induced nephrosisand reverses the profound decrease in renal blood flow normally observedin the acute phase of this disease. Parallel experiments with marrowirradiation demonstrated that the predominant effect of EFA-deficiencyon preserving renal blood flow and glomerular filtration could beattributed to on blocking mononuclear migration into the interstitium,Harris, K., et al 1990. J. Clin. Invest. 86: 1115.

Importantly, this effect is not confined to the kidney. EFA-deficiencyinhibits the development of autoimmune insulitis in mice receiving lowdose streptozotocin and in the diabetes-prone BB/Wor rate, Wright, J.,et al 1988. Proc. Natl. Acad. Sci. 85: 6137; Lefkowith, J., et al 1989.J. Exp. Med. 161: 729. Both are models of diabetes in which insulitis ispreceded by islet infiltration by monocytes. It has been demonstratedthat pancreatic islets undergoing oxidative stress after in vivoexposure to streptozotocin release an uncharacterized lipidchemoattractant, specific for monocytes, resembling from that releasedby isolated glomeruli, Muir, A., et al 1991. Diabetes 40: 1459. In skingraft experiments, we have observed impaired wound healing in fatty aciddeficient animals with marked inhibition of monocyte accumulation in thetraumatized skin and inhibited formation of granulation tissue(unpublished observations). EFA-deficiency has previously been shown toprotect against atherosclerosis, Holman, R. T. 1960. J. Nuir. 70: 405,and inhibit carrageenan-granuloma formation, Bonta, F., et al 1977.Prostaglandins 14: 295, and diminish the leukocyte inflammationassociated with experimental myocardial infarction.

The release of a potent uncharacterized chemoattractant for monocytes,Rovin, B., et al 1990. J. Immunol 145: 1238, has been described inshort-term cultures of nephritic glomeruli from rats on a standard diet.Production of this chemoattractant is markedly enhanced after inductionof nephritis. EFA-deficient nephritic glomeruli do not release themonocyte chemoattractant. In vivo studies employing inhibitors ofcyclooxygenase and lipoxygenase have demonstrated that thischemoattractant is not a product of either pathway. Administration of aplatelet activating factor (PAF) receptor antagonist similarly failed toinhibit the glomerular influx of macrophage. Lipid (Blight-Dyer)extraction of nephritic glomeruli from control diet animals has yieldedchemoattractant activity in the organic phase, Rovin, B., et al 1990. J.Immunol 145: 1238.

These findings suggest that a lipid pathway, metabolically linked todietary fatty acids, may provide a generalized mechanism for theinduction of monocyte infiltration into tissues.

An article by Graziano Guella et al., Helvetica Chimica. Acta. 70;1050-1059 (1987) describes various long chain acetylenic enol ethers ofglycerol derived from marine sponges. The compounds identified, however,are not the same as the lipid chemoattractant of this invention becausethe compounds described in Guella et al. have properties inconsistentwith the properties of the compound of this invention. Specifically, theactivity of the Guella et al. compounds, if any, would not be inhibitedby selective reducing agents as sodium borohydride. Likewise, thecompounds of Guella et al., which are ethers of marine sponges, havenever been isolated and purified from urine or plasma of mammalsincluding humans. Furthermore, unlike the lipid chemoattractant of thisinvention, the Guella et al. compounds would not lose their activity, ifthey exhibit any, at temperatures in excess of 180° C. Finally, theGuella et al. compounds include conjugated double bonds which exhibit acharacteristic UV absorption (λ_(max)) of 292. The lipid chemoattractantof this invention does not exhibit UV absorption at 292, indicating thatthe double bonds are not conjugated.

SUMMARY OF THE INVENTION

This invention encompasses a substantially homogeneous lipidchemoattractant released from stressed mammalian tissue which is aneutral lipid which is acid labile and stable to base and is stable inboiling water. This lipid recruits macrophages but not neutrophils tostressed tissue. The invention further encompasses a method fordetecting injured tissue by detecting the presence of the abovedescribed lipid chemoattractant in body fluids such as urine, serum andsaliva. The invention also includes a method for reducing recruitment ofmacrophage to injured tissue by reducing the amount of the above lipidchemoattractant or by blocking the interaction of this lipidchemoattractant with monocyte/macrophage binding sites. The addition ofthis lipid chemoattractant to injured skin tissue promotes healing.

A purification scheme has been devised that results in thechromatographic isolation of an almost homogeneous neutral lipid thatconstitutes the newly described factor of chronic inflammation of thisinvention. The purification scheme includes extracting a crudeorganically extracted medium containing the lipid chemoattractant andcontaminants from a mammalian source. The extract is separated bysilica-based separation techniques to give a first separation productcomprising the lipid chemoattractant and at least one fatty acidcontaminant. The separation product is hydrolyzed with an alkaline agentto give a hydrolyzed product comprising the lipid chemoattractant andcleaved fatty acid contaminants. Finally, the hydrolyzed product isseparated using silica-based separation techniques to give anessentially pure solution of the lipid chemoattractant.

The isolated compound does not resemble any previously described lipidinflammatory factor. It specifically induces the chemotactic migrationof monocytes into extravascular tissues. It has been isolated from bothanimal and human examples of organ disease associated with theaccumulation of monocytes/macrophages on a persistent basis. It has beenshown to be associated with the following conditions:glomerulonephritis, nephrosclerosis, interstitial nephritis, nephrosis,and diabetes. The factor is inferentially associated withatherosclerosis, pneumonitis, pulmonary fibrosis, myocardial infarctionand fibrosis, and chronic dermatitis.

The factor itself resembles, based on its chemical properties, amonoglyceride with a vinyl ether linkage. As an agonist it could be usedas an aid to wound healing. Antagonism of this factor could be expectedto suppress chronic inflammation of the lungs, heart, blood vessels,pancreas, kidney, peripheral vasculature and the kidney.

Detection of this material has occurred in the urine and peritonealfluid of patients with a variety of chronic kidney diseases. Detectionof this factor is a useful diagnostic or prognostic assay in the urine,blood, peritoneal fluids, or stool of patients with myocarditis,myocardial infarction, pneumonitis, pulmonary fibrosis,glomerulonephritis, interstitial nephritis, nephrosis, progressive renalinsufficiency, atherosclerosis peripheral vascular disease, chronicdermatitis or inflammatory bowel disease. The use of a blood diagnosticassay may be of particular utility in the diagnosis and/or management ofatherosclerosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. HPLC chromatographic isolation of a peak of chemotactic activityisolated from inflamed renal glomeruli. The activity is designated asinducing cell migration, expressed as cells (monocytes)/HPF.

FIG. 2. The chemotactic response of neutrophils monocytes, andmacrophages to chromatographically purified lipid chemoattractant.

FIG. 3. HPLC chromatographic isolation of a peak of chemotactic activityisolated from supernatants fed albumin replete with fatty acids, noalbumin (control) and urine from animals rendered proteinuric byadministration of 1 gram albumin intraperitoneally for 5 days.

FIG. 4. Comigration of lipid chemoattractant with monocylglycerols, asisolated from medium of proximal tubules fed albumin oleate.

FIG. 5. HPLC isolation of a single peak of lipid (as determined by UVabsorption scan between 200 and 250 nM) possessing chemotactic activity.

FIG. 6. Plot indicating the presence of lipid chemoattractant in plasmaof humans suffering from atherosclerosis, ("PATIENT"), and the relativeabsence of the lipid chemoattractant in the plasma of healthy humans,("CONTROL").

DETAILED DESCRIPTION OF THE INVENTION

This invention is a purified lipid chemoattractant derived from stressedmammalian cells as well as a method for purifying the lipidchemoattractant. Furthermore, this invention includes inhibitors of thelipid chemoattractant as well as inhibitors of monocyte chemotaxis.

The purified lipid chemoattractant of this invention is useful fordeveloping methods and agents for detecting the lipid chemoattractant instressed mammalian cells. The purified agent is also useful fordeveloping and identifying inhibitors of the agent as well as inhibitorsof monocyte chemotaxis, the physiological mechanism that produces theagent. The purified lipid chemoattractant of this invention can also beused topically or internally to promote wound healing.

The isolation and purification of the lipid chemoattractant of thisinvention has permitted us to study and characterize the agent based onit chemical and biological characteristics as well as its structure. Ourunderstanding of the chemical structure of the lipid chemoattractant ofthis invention is based on a number of types of studies performed on theagent including refining and improving methods for purifying the agent,characterizing biochemical properties of the agent, characterizing themetabolic pathway to synthesis of the agent, identifying inhibitors tothe synthesis of the agent, by identifying inhibitors of monocytechemotaxes and by preparing synthetic analogs of the agent. None ofthese characterization efforts nor the identification of inhibitionswould have been possible without first having a substantiallyhomogeneous form of the lipid chemoattractant to work with.

A purification method that reproducibly yields a substantiallyhomogeneous monocyte chemotactic factor released by glomeruli or renalinterstitium has also been developed. Previous attempts atcharacterizing this factor were difficult because isolated fractionspossessing biological activity were extremely heterogeneous andinsufficient purity precluded biochemical definition of the lipidchemoattractant.

The identification of this novel factor as a neutral lipid and itsunusual stability to alkaline methanol has allowed further refinement inits isolation such that we can prepare the lipid factor, with potentchemotactic activity, with minimal contamination by other lipids fromboth tissue conditioned medium and bodily fluids such as urine.Unpurified sources of the lipid chemoattractant include, but are notlimited to the mouse monocyte leukemia line, RAW 264.7 is used as anindicator. The cell line is distinctive for its normal responsiveness tophysiological concentrations of macrophage activation signals, includingendotoxin and gamma interferon. These cells have been used as a modelsystem for evaluating monocyte chemotaxis. These cells are periodicallycompared to glycogen-elicited rat peritoneal macrophage to assure theirreliability.

In a standard protocol for eliciting the release of a chemotacticfactor, rats are injected with rabbit anti-rat glomerular basementmembrane antibody. Control rats received rabbit immunoglobulin preparedfrom non-immune serum. The glomeruli are harvested 12 hrs. later aftersaline-perfusion of the kidney and cultured in RPMI 1640 with 10 mMHEPES and 0.25% fatty acid free albumin. After 2 hours, the glomeruliand medium are extracted by the method of Bligh and Dyer or, in apreferred step, with ethyl acetate. Aliquots tested in micro chemotaxischambers, according to the method of Falk, Falk, W., et al 1980. J.Immunol. Meth. 33: 239, demonstrate chemotactic activity as measured bythe enumeration of cells migrating through filters. Parallel extractionsof normal glomeruli and EFA-deficient glomeruli yield no activity.

We have identified numerous physical, chemical, and biologicalproperties for this novel neutral lipid macrophage chemoattractant.

The factor is insensitive to trypsin, pronase, or collagenase.Inhibitors of cyclo-oxygenase and lipoxygenase activity exert no effecton the generation of the chemo lipid in vivo or in vitro, but conditionsfavoring oxidation increase its activity. It is not inactivated byheating up to 100° C. for five minutes. It cannot be modified bydiazomethane indicating the lack of a free carboxy group. The factor canbe sililated with trimethylsililate (BSTSMFA), delaying its elution onHPLC, with recovery of biological activity after hydrolysis in anaqueous medium. This suggests the presence of free hydroxyl or aminogroups. It is extracted into ethyl acetate and migrates as a neutrallipid by TLC and HPLC chromatographic techniques. Exposing the materialto 0.5 N KOH in methanol results in no loss of chemotactic activity.However, subjecting the material to 0.5 N HCL in methanol for 30 minuteseffected virtually complete loss of detectable chemotactic activity asdid exposure to concentrated HCL fumes, indicating that the biologicalactive molecule has a vinyl ether or similarly acid labile linkage, andthat an ester bond is not necessary for biological activity. Lipidphosphorus is not detected on either the TLC fraction or the active HPLCfractions either by phosphomolybdate spray (TLC) or by ashing (HPLC/TLC)and assaying by the method of Rouse et al., Rouse, R., Fleischman, B.,and Yamamoto, F. 1970. Lipids 49: 497. It is sensitive to reduction byvitride or sodium borohydride. This factor has no chemotactic activityfor polymorphonuclear leukocytes (FIG. 2). According to all thin layerand column-based chromatographic analyses, the lipid chemoattractant ofthis invention is a short-chain (n(12) monoalkyl glycerol or an oxidizedlong-chain (n)16) monoalkylglycerol. In summary, its chromatographicproperties as an acid sensitive, non-polar lipid with no effect onneutrophils distinguishes it from platelet activating factor and LTB₄(FIG. 1), the only other defined lipid macrophage chemotactic factors,and identify the isolated factor as novel.

It has been demonstrated that the proximal tubules are a source of thechemotactic lipid by isolating proximal tubules by density sedimentationof renal cortical digests from albumin-injected rats. When proximaltubules were placed in overnight culture, lipid extracts of harvestedsupernatants demonstrated release of considerable chemotactic activity.The elution characteristics on HPLC were identical to those describedabove. On the other hand, extracts of supernatant from the cultureproximal tubules from control rats were inactive.

Chemoattractant activity can be stimulated in proximal tubules fromnormal rats placed in overnight culture in medium containing 5 mg/ml oflipid replete bovine serum albumin (BSA). Supernatant from the tubulescultured in the presence of lipid-replete albumin contained chemotacticactivity similar in magnitude to that seen in the supernatants oftubules from the protein overloaded animals. However, the supernatantsfrom the tubules cultures in the presence of lipid-depleted albumincontained little activity. The isolated activity from the lipid-repletedBSA supernatants showed mobility identical to that of the activity inthe urine of proteinuric rats on TLC. The elution profile of chemotacticactivity recovered from proximal tubules on HPLC was superimposable onthat observed in the urine of proteinuric animals. (FIG. 3) Feeding theproximal tubules albumin that has been selectively loaded withindividual fatty acids has demonstrated that oleate and the myristateare the most stimulatory fatty acids.

Using the system of extraction into ethyl acetate, separation by TLC andpurification by reverse phase HPLC as described in Examples I and IIbelow, we have identified a molecule with identical chromatographicproperties in the urine of patients with proteinuric kidney disease. Wehave also identified the molecule in the plasma of humans withatherosclerosis and the relative absence of the molecule in patientswithout the condition (FIG. 6). Conditions in which high levels of thislipid chemoattractant has been identified include diabetes, membranousnephropathy, focal segmented sclerosis and glomerulonephritis. Twopatients with proteinuria and stable renal function have control levelsof this agent. These data indicate that the presence of this material inurine and other fluids has diagnostic and prognostic utility fordetecting tissue injury such as the kidney. (Table I).

The lipid chemoattractant is detected mammalian tissues and serumsources by subjecting the tissues and serum or any other potentialsource of lipid chemoattractant to the purification methods described inExamples II and III. The resulting pooled product then undergoesstandard bioassay testing to determine whether or not the lipidchemoattractant is present and also to determine the amount present.

The lipid chemoattractant has additional distinctive attributes. It isnot sequence or species specific. The same factor, as defined bychromatographic characteristics and biological activity, has beenisolated from humans, rats, and mice. Monocytes from any of thesespecies are equally responsive to the factor regardless of the speciesfrom which it is isolated. Cells or organs known to produce this factorinclude vascular smooth muscle cells, pancreatic islets, renalglomeruli, renal proximal tubular epithelium, and intact aortas. Signalsor stresses that we have shown to promote release of this factor includeuptake of lipoproteins, including albumin, intracellular oxidation,anoxia, tissue culture, immune complexes, and mechanical stretch ofluminal structures, as is seen in obstruction of hypertension.

                  TABLE I    ______________________________________    Monocyte CTX Activity in Extracted,    Chromoatographed Urine (18'-20')                   MONOCYTE                   MIGRATION                   (cells/filter)    ______________________________________    PROGRESSIVE    RENAL    DISEASE    FSGS             11,039    FSGS             23,140    IgA/TIN          24,386    Diabetes         21,184    STABLE RENAL    FUNCTION    Control           2,492    FSGS (obese)      2,552    FSGS (non-progressive)                      2,492    ______________________________________

The identification of the lipid chemotaxis factor as a vinyl ethermonoalkyl glycerol has permitted the design of inhibitors. A variety ofnaturally occurring fatty acids were screened for the capacity toinhibit the oleate stimulation of the novel lipid chemoattractant.

Inhibitors of the synthesis of the lipid chemotaxis factor include longchain polyunsaturated fatty acids and particularly C18:3, C:20:3, andC:20:5 fatty acids. C18 fatty acids with substitutions between C₆ -C₁₂,e.g., ricinoleate, are also inhibitory. The most inhibitory fatty acidsreflect modifications of fatty acids between the C9 and the omegacarbon. In order of potency, the fatty acid inhibitors are: ricinoleicacid>eicosopentanoic acid>mead acid>docosohexanoicacid>linolenate>eicosadienoic acid. Ricinoleic acid is 100% inhibitoryat micromolar concentrations.

Ethanol is inhibitory as are lipid soluble antioxidants such asethoxyquin, butylated hydroxytoluene, and butylated hydroxyanisole.Cytrochromic P₄₅₀ enzyme inhibitors such as ketoconazole andclotrimazole are also inhibitors of the synthesis of the lipidchemoattractant as are alcohols of long chain fatty acids.

A prototype receptor antagonist has been synthesized from commerciallyavailable beef heart phosphotidylcholine, which contains 30%plasmalogens (Sigma Chemical Co., St. Louis, Mo.). Thephosphotidylcholine/plasmalogen mixture was subjected to phospholipase Cexposure followed by alkaline hydrolysis in 0.5 NaOH in methanol and TLCseparation. This yielded a mono-alkyl vinyl ether of glycerol, which waslabile in 0.5 N HCl. Its two principal substituents at the SN-1 positionwere the corresponding vinyl ether derivatives of palmitate and oleate.This mixture had no intrinsic chemoattractant agonist activity. However,at 10⁻⁶ M concentrations, it completely blocked monocyte migration invitro to biologically active fractions prepared from chromatographicisolates from proteinuric urine. This demonstrates that monoglycerideswith long chain fatty acid-derived substituents linked as vinyl ethersin the Sn-1 position of glycerol inhibit the leukocyte response tobiologically active mono-alkyl glycerides.

Other monocytechemotaxis inhibitors include inhibitors lysomalacidification such as chloroquine and ammonium chloride.

Thus the lipid chemoattractant of this invention has the generalstructure I: ##STR1## where R₁ is a C4-C22 alkyl group optionallycontaining 1-3 branched group. Each branched group may be a hydroxylgroup, oxygen, or a carbonyl group. R₁ may include 1-3 unsaturateddouble or triple bonds but R₁ is unconjugated so none of the double canshare adjacent carbon atoms.

R₂ may be oxygen (O) or HOH. An important aspect of the lipidchemoattractant is that it includes at least one oxygen or carbonylgroup. If R₂ is oxygen, then R₁ may, but need not include a carbonylgroup or an oxygen group that is not part of a hydroxyl group. If R₂ isHOH, the R₁ must include an oxygen that is not part of a hydroxyl group,or carbonyl group as part of the alkyl backbone, or as a branched group.

In addition, those skilled in the pharmaceutical arts will recognizethat interfering with the production of I through interference with thepathway that produces I will effectively reduce macrophage migration.

EXAMPLE I

The lipid chemoattractant of this invention has been purified by thefollowing procedure:

Approximately 20-30 ml of urine or medium conditioned by proximaltubules fed albumin complexed with fatty acids are extracted with ethylacetate (1:1. v/v). After phase separation via centrifugation, theorganic phase is collected and subsequently dried using a Buchiirotatory evaporator. The dried samples are stored under argon in asilylated vial.

A 2-step TLC procedure is used to isolate fractions of the extractedurine sample with corresponding chemotactic activity. Samples arespotted on silica gel plate (Whatman LK6DF plate with a pre-absorptionzone) and developed initially with a solvent system ofchloroform:methanol: acetic acid (60:25:1), v/v/v) to an R_(f) of 0.3.The plate is dried and then exposed to a second solvent system ofpetroleum ether: ether: and acetic acid (80:20:1) and developed to anR_(f) of 1.0. All chemotactic activity elutes from an area comigratingwith short and long-chain monoglycerols, R_(f) =0.25±0.05 (FIG. 4), anarea which was scraped and eluted with chloroform:methanol (1:1, v/v).

To eliminate contaminating lipid esters which predominate at this pointand allow subsequent chromatographic purification to near homogeneity,we utilized the surprising base stability of the lipid chemoattractantto permit substantial enrichment of bioactivity. Thus, the dried eluentsare subjected to mild alkaline hydrolysis (0.5N KOH in methanol, at 37°C. for 30 min.). After the completion of the hydrolytic process, theproducts are extracted with ethyl acetate and separated from theresulting fatty acids with HPTLC (Whatman LHP-KDF with a pre-absorptionzone) using a solvent system of petroleum either:ether:isopropanol(3:2:0.6, v/v/v) to an R_(f) of 1.0 followed by a 5 min overdevelopment. The fraction with corresponding chemotactic activity,between R_(f) 0.4-0.8, is scraped and eluted with chloroform:methanol1:1, v/v and subjected to further purification using HPLC.

The fraction with chemotactic activity is further purified by HPLC. Thesample is loaded onto a Supelco LC-DP diphenyl reversed phase HPLCcolumn (5 u, 25 cm×4.6 mm) with the elution solvent delivered via aWaters liquid chromatograph system equipped with a model 600E controllerdriven by the Millennium v 1.1 data acquisition and analysis software,and monitored with a Waters model 996 photodiode array detector. Agradient solvent system of acetonitrile (ACN) and water is used toseparate the chemoattractant from other contaminants. Initially thesolvent system ratio is ACN:H₂ O (25%:75%) for 10 min, after which theACN level is raised to 60% in a duration of 5 min, and to 80% in periodof 2 min. Finally, the ACN % was raised to 100% in 10 min. Fractionswere collected in 30 second intervals using a Waters fraction collector.All chemotactic activity elutes between 23 and 24 minutes at an ACN % of92% and the chromatogram shows a single absorbance peak overlapping thechemotactic activity (FIG. 5).

The lipid chemoattractant purified by the method of Example I exhibitsthe following properties:

                  TABLE II    ______________________________________    Properties of the homogeneous lipid chemoattractant*    ______________________________________    (a)    acid           labile    (b)    base           stable    (c)    100° C. in water                          stable    (d)    macrophages    recruits    (e)    neutrophils    not-recruited    (f)    injured tissue secreted by    (g)    chemical structure                          Sn-1 vinyl ether of glycerol    (h)    lacks phosphorous    (i)    heat lability  activity destroyed 180° C.    ______________________________________     *These properties were demonstrated as follows:     (a) Placed in 0.5N HCl in methanol per 30' at 37° C.     (b) Placed in 0.5N KOH in methanol for 30' at 37° C.     (c) Boilcd in H.sub.2 O at 100° C. for 15'.     (d) d + e = side by side comparison of the migration of neutrophils     (elicited by the installation of zymosanactivated serum in the rat     peritoneum) and monocytes (isolated by density sedimentation from the     peripheral blood of rats and humans) and macrophages (isolated by     instillation of heatinactivated fetal calf serum in the rat peritoneum).     (h) Lack of phosphorous established by molybdate spray on thin layer     chromatograms and by ashing analysis.     (i) Dried to film by evaporation under nitrogen and heated in an oven at     180° C. for thirty minutes.

                  TABLE III    ______________________________________    Chemical Derivatisations Of The    Homogeneous Lipid chemoattractant**    AGENT             STRUCTURAL INDICATION    ______________________________________    (a)   Sodium Borohydride                          Carbonyl group                           ##STR2##    (b)   Periodate       Adjacent alcohols on adjacent                          carbonyl alcohol groups.                           ##STR3##                          or                           ##STR4##    (c)   Acetic Anhydride                          Alcohol group                          R--C--OH    (d)   Acid            Vinyl ether COCCC                          or                          epoxy group                           ##STR5##     **These structural indications were demonstrated as follows:     (a) The lipid chemoattractant is dissolved in ethanol and exposed to soli     NaBH.sub.4 overnight at room temperature. The material is then reextracte     with diethylether in water and tested for biological activity.     (b) Lipid chemoattractant is dissolved in 1% periodate (w/v) in sodium     acetate buffer for 1 hour at room temperature after which it is     reextracted in ethyl acetate and tested for biological activity.     (c) Lipid chemoattractant is suspended in acetonitrile and exposed to     acetic acid anhydride and pyridine for 16 hours at room temperature.     Acetylation is reversible by placing acetylated chemoattractant in 0.5 N     KOH in methanol for 30 minutes at 37° C.     (d) lipid chemoattractant is placed in 0.5 N HCl in methanol for 30     minutes at room temperature.

All of the above derivitisations destroy biological activity and alterthe chromatographic properties of radiolabeled lipid chemoattractant.

EXAMPLE II

This Example describes an improved and alternate method for preparing aessentially homogeneous solution of the lipid chemoattractant of thisinvention.

The lipid chemoattractant agent is extracted into ethyl acetate fromconditioned medium generated from a variety of mammalian sourcesincluding human sources such as rat renal proximal tubules or vascularsmooth muscle cells or human renal proximal tubules with albumen oleateor lipid containing serum. Other fluid sources of the lipidchemoattractant include plasma, serum, urine, and cyst fluid from humanand animal sources. In addition to the lipid chemoattractant, the mediumcontains impurities and contaminants such as monoalkylglycerols andother low molecular weight compounds (M.W. less than 1000) that do notexhibit biological activity.

The first extract in eluted over a silica column in a mobile phase ofpetroleum ether:ether (25:75 v/v).

The eluted extract is subjected to alkaline hydrolysis 0.5N KOH inmethanol, at 37° C. for thirty minutes and is re-eluted over the silicacolumn in the same manner as above to provide a second extract.

The second extract is then concentrated by evaporation under nitrogenand separated by passage over normal phase, Supelco silica column (5 μ;4.6×250 mm) via a Waters liquid chromatograph system describe in ExampleI. A solvent system of hexane: methyl-t-butyl ether:isopropanol(60:40:10 v/v/v) is used to separate the lipid chemoattractant for otherremaining contaminants. The lipid chemoattractant elutes at 18-20minutes.

The concentrated lipid chemoattractant is now essentially pure and itmay be pooled and used for a variety of purposes. Further purificationof the lipid chemoattractant is still possible, however and is achievedby first preparing a third extract by the active fraction by undernitrogen, resuspending it in acetonitrile, and elution by passage of aSupelco LC-DP dipheny reversed phase HPLC column delivered via a Watersliquid chromatograph system as described in Example I. The same gradientof solvent (acetonitrile:water) is used as is described in Example Iwith the exception of the addition of 50 nM ammonium acetate.

The third extract is applied to a reversed phase C-18 guard column in25% acetonitrile/75% H₂ O. The column is then equilibrated in 100% waterand the activity is then eluted in 100% acetonitrile moving a 1ml/minute.

The lipid chemoattractant is further purified by loading onto Phenomenexcyanaopropyl column (3 μ, 3 mm×150 mm) in hexane: methyl t-butyl ether(60:40 v/v), 1 ml/mm, with elution time of the activity being 3-5minutes. These subsequent purification steps takes the essentially puresolution of lipid chemoattractant and results in a nearly homogeneouspreparation of lipid chemoattractant as determined by NMR and MStesting.

EXAMPLE III

The sensitivity of the lipid chemoattractant to borohydride indicatesthe presence of a carbonyl group, which distinguishes it from long chainacetylenic enol ethers of glycerol. The presence of a carbonyl group onthe backbone of the structure is supported by the observation that C¹⁴-labeled dihydroxyacetone (DHA) is incorporated into the lipidchemoattractant. The 2(C) labeled C¹⁴ -DHA incorporation into the lipidchemoattractant has been demonstrated in two types of experiments. Inthe intact cell assay outlined in Example I, C¹⁴ -DHA is (100 μM)included with albumen-oleate in the medium of rat proximal renal tubulesplaced in short-term tissue culture where it is incorporated into thelipid chemoattractant. The labeled chemoattractant was then purified bythe method detailed in Example II, and each fraction that demonstratedbiological activity also demonstrated incorporation of C¹⁴ -DHA.Analogous results are observed in a cell free lipidchemoattractant-generating system consisting of 100 μM DHA, 50 μM oleylalcohol, 10 MM ATF, 4 mM MgCl₂, 20 mM NADH, 33 mg/ml catalase, and 500μg of microsomal protein from rat kidney cortex. Incubation of thismixture in Kreb's-Henseleit medium for one hour at 37° C. yields a lipidchemoattractant with DHA incorporation that is chromatographically andbiochemically identical to that of the lipid chemoattractant isolatedfrom intact kidney cells or proteinuric urine.

What is claimed is:
 1. A method for detecting the presence of injuredtissue in a human comprising collecting body fluid from a human,preparing an essentially pure lipid chemoattractant, and testing theresulting essentially pure chemoattractant product in a standardbioassay wherein the lipid chemoattractant has the following structure:##STR6## where R₁ is a C₄ -C₂₂ alkyl group containing from 1 to 3branched groups selected from the groups consisting of hydroxyl, oxygen,or carbonyl, and from 1 to 3 unconjugated double or triple bonds,whereinR₂ is ═O, or --OH, and wherein R₁ and R₂ must include at least onecarbonyl group or oxygen that is not part of a hydroxyl group.
 2. Themethod of claim 1 wherein the an essentially pure lipid chemoattractantis prepared from the human body fluid by the steps comprising:a.separating the body fluid using a silica-based separation technique togive a first separation product comprising the lipid chemoattractant andat least one fatty acid contaminant; b. hydrolyzing the first separationproduct with an alkaline agent to give a hydrolyzed product comprisingthe lipid chemoattractant and cleaved fatty acid contaminants; and c.separating the lipid chemoattractant from the hydrolyzed product usingsilica-based separation techniques to give a second separation productcomprising an essentially pure lipid chemoattractant.
 3. The method ofclaim 1 wherein the injury is atherosclerosis and the body fluid isplasma.
 4. The method of claim 1 wherein the injury is chronic kidneydisease and the body fluid is selected from the group consisting ofplasma and urine.